A chemical synthesis of useful substances such as a protein or vitamin requires complicated processes, and thus is expensive. Therefore, efficient processes for producing such substances by fermentation methods using microorganisms have been developed.
Biotin is a vitamin, essential for animals, plants and microorganisms, and can be used as an additive for a feed or a liquid for transfusion, or the like. The development of such fermentation methods for biotin has been attempted, and Japanese Unexamined Patent Publication (Kokai) No. 61-202686 and No. 62-155081, and WO87/01391 disclose processes for preparing biotin using strains of Escherichia coli improved by a genetic engineering technique.
Generally speaking, when the substance is produced using a microorganism, it is an effective means of achieving a high density of the microorganism in a culture broth with a fed-batch culture or the like, in view of not only an increased yield of the product but also an efficient employment of a fermenter and an efficient recovery of the product. In the case of Escherichia coli, many advantages could be obtained from the high density cultivation, particularly of Escherichia coli improved by a genetic engineering technique. But, in the case of Escherichia coli, acetate is formed with a metabolism of nutrients, and accumulated in a considerable amount in the culture broth, thereby inhibiting an activity of growth of the cell, and therefore, the realization of a high density cultivation was difficult. As the means of solving the above problem and achieving a high density cultivation, a dialysis or filtration cultivating method is known wherein the formed acetate is removed from the cultivation system by dialysis or filtration of the culture broth.
Further, a pressurized culture was proposed wherein a pure oxygen gas is used to maintain a high concentration of a dissolved oxygen in the culture broth, thereby suppressing the amount of acetate formed (Matsui, et al, Synopses of Presentations on Convention in 1986, Fermentation Engineering Association of Japan, p. 206).
Nevertheless, problems arise in the dialysis or filtration culture, for example, the necessity for exclusive use equipment or a complicated maintenance of the equipment, and thus this is not economical or practical. Further, some useful substances (e.g., biotin) produced by Escherichia coli and accumulated can be dialyzed or filtrated from the culture broth together with acetate and therefore, it is necessary in the recovery step of the useful substances to deal with a large amount of the liquid containing a low concentration of the substances, and thus the advantage gained by carrying out the high density cultivation is almost completely lost. The high density cultivation of Escherichia coli with the pressurized culture brings a complicated control during cultivation operations or conditions, and is not easily achieved.